CN102994647A

CN102994647A - Typing and quantitative detection method and kit for human papilloma virus (HPV)

Info

Publication number: CN102994647A
Application number: CN2012102065641A
Authority: CN
Inventors: 吴勇; 孙婷婷
Original assignee: NINGBO HEALTH GENE TECHNOLOGIES Co Ltd
Current assignee: Ningbo Haier Shi Gene Technology Co.,Ltd.
Priority date: 2012-06-21
Filing date: 2012-06-21
Publication date: 2013-03-27
Anticipated expiration: 2032-06-21
Also published as: US20150140547A1; US9994922B2; CN102994647B; WO2013189306A1

Abstract

The invention discloses a typing and quantitative detection method for human papilloma virus (HPV). The typing and quantitative detection method comprises the following steps: (1) collecting a cervical sample; (2) extracting DNA (deoxyribonucleic acid) from the sample; (3) performing PCR (polymerase chain reaction) by taking the extracted DNA sample as a template; and (4) separating the sample through a capillary electrophoresis method. The typing and quantitative detection method disclosed by the invention has the advantages of high sensitivity, good repeatability, strong accuracy, strong flexibility and low cost.

Description

Somatotype quantitative detecting method and the test kit of a kind of human papillomavirus (HPV)

Technical field

The present invention relates to a kind of detection method, somatotype quantitative detecting method and the test kit of especially a kind of highly sensitive, good reproducibility, accuracy is strong, handiness is strong, cost is low human papillomavirus (HPV).

Background technology

Human papillomavirus (Human Papillomavirus is called for short HPV) is a kind of epithelium venereal disease poison of having a liking for, and the specificity of height is arranged.Strong and weak according to virulence, HPV is divided into two kinds of high-risk-type and low risks, and whether " can be carcinogenic " be the item key of risk level size.The new cases of the annual cervical cancer in the whole world are nearly 500,000, are the second largest malignant tumours of threat women life and health except mammary cancer.The nosetiology relation of HPV and cervical cancer is clear and definite, and infecting cervical cancer from HPV needs the long period, by detecting to understand whether infect HPV, early finds early treatment, thereby effectively prevents the generation of cervical cancer during this period.

At present main technological method is as follows: 1. in situ hybridization: advantage is that specificity is good; Shortcoming is a large amount of purer DNA of complex operation much time power, needs.2.DNA direct trapping: advantage is the application that a large amount of this technology of Data support is arranged, and can detect high-risk type; Shortcoming is that sensitivity is lower than PCR; Can not determine concrete HPV hypotype.3. hypospecificity PCR: advantage is that specificity is better; Shortcoming is that labour intensity is large.4. universal primer PCR: advantage is highly sensitive, and multiple hypotype once can increase; Shortcoming is to distinguish the hypotype of determining concrete HPV.5.PCR product direct Sequencing: advantage is to be particularly useful when the HPV Subtypes of determining not yet to know at present and mutation research; Whether very sensitive, particularly all the more so to the clinical sample of HPV polyinfection shortcoming is, should not be as the in-vitro diagnosis method.6.PCR-enzyme-linked immunosorbent assay: advantage is highly sensitive; Shortcoming is that sample consumption is large.7.PCR-solid phase reverse hybridization: advantage is that efficient is high, once experiment can be told a plurality of HPV hypotypes; Shortcoming is that repeatability is relatively poor.8. quantitative fluorescent PCR: advantage is highly sensitive, easy to operate, consuming time weak point; Shortcoming be flux few, once can only detect 1-2 hypotype and definite somatotype.9. advantage is that cost is relatively low, and is simple to operate; Shortcoming is to observe the variation of cellular form, but can not determine the cause of disease that the cellular form variation occurs; The position of sampling determines the result of detection.

To sum up, the shortcoming that there is complex operation much time power in the main detection method of present human papillomavirus (HPV), sensitivity is low, labour intensity is large, sample consumption large, repeatability is relatively poor, flux is few.

Summary of the invention

The somatotype quantitative detecting method that the purpose of this invention is to provide a kind of highly sensitive, good reproducibility, accuracy is strong, handiness is strong, cost is low human papillomavirus (HPV).

The present invention adopts following technical scheme:

One aspect of the present invention relates to the somatotype quantitative detecting method of a kind of human papillomavirus (HPV), comprises the steps: (1) collection uterine neck sample; (2) sample DNA extracts; (3) carrying out PCR take the DNA sample that extracts as template reacts; (4) with the method sample separation of electrocapillary phoresis.

Preferably, described step (2) comprises following substep:

1) get 500 μ L cell-preservation liquids in 1.5mL EP pipe, centrifugal 5 minutes of 13000g removes supernatant liquor, and the bottom is cervical exfoliated cell;

2) in the EP pipe that contains cervical exfoliated cell, add 500 μ L lysates, cracking 10 minutes;

3) in lysate, add the free DNA of 100 μ L magnetic beads absorption;

4) with scavenging solution magnetic bead is cleaned twice;

5) 100 μ L elutriant eluted dnas.

Preferably, described step (3) comprises the substep that carries out in proportion thermal cycle reaction after sample panel adds reagent and sample and mixing by certain temperature.

Preferably, the thermal cycling temperature in the substep of described step (3) is respectively 94 ℃, 60 ℃, 70 ℃, 4 ℃.

Preferably, described step (4) comprises preparation GeXP genetic analyzer sample, prepares the step of parting liquid, electrocapillary phoresis sample separation, interpretation of result.

Another aspect of the present invention relates to a kind of test kit for aforesaid method, comprises following reagent: primer pipe, solution X, PCR buffer reagent, 25mM magnesium chloride, polysaccharase and positive control.

Beneficial effect of the present invention is:

1. highly sensitive, good reproducibility: adopt laser induced fluorescence(LIF)-PMT, have hypersensitivity.

2. accuracy is strong: GeXP adopts capillary electrophoresis that the PCR product is carried out separation detection, non-specific amplification product, primer dimer and specific amplification products can be separated, at utmost reduce false positive.

3. high-throughput: native system adopts two (96 hole) plates, automatic sample and sample tracer technique, realize a single reaction detection 30-40 site, can do simultaneously 192 reactions (such as 192 patient's samples, 19 kinds of Respiroviruses of each sample detection, 22 sites), went out the result in one day; For co-infected patients, present method can disposablely provide accurate report, avoids undetected.

4. accurate quantification: but accurate quantification pathogen gene copy number.

5. handiness is strong: can increase according to demand at any time or delete the HPV hypotype.

6. cost is low: be beneficial to large-scale promotion.

Embodiment

The below is described in further detail the present invention according to embodiment.

The present invention has founded 25 kinds of HPV hypotypes of a kind of disposable detection, comprise 6 kinds of low risks 6,11,42,43,44,81 and 19 kind of high-risk HPV hypotype 16,18,26,31,33,35,39,45,51,52,53,56,58,59,66,68,73,82,83 somatotype detection by quantitative scheme, concrete hypotype sees Table 1.

The Auele Specific Primer of 25 kinds of HPV hypotypes of the present invention is for the oncogene E6/E7 on each subtype gene group design, and the method by multiplex PCR and electrocapillary phoresis again distinguishes each hypotype according to the difference of expanding fragment length.

Set up the contrast (table 1) of reliable sample quality and reaction process

A) the contrast confidential reference items β-globin of people DNA integrity: guarantee in checkout procedure false negative is avoided in the judgement of sample quality.

B) normal reaction contrast confidential reference items pcDNA3.1 (+): monitoring PCR reaction efficiency, avoid false negative.

2. by artificial adding reaction confidential reference items pcDNA3.1 (+) copy number of HPV hypotype is carried out quantitatively.

3.25 plant the primer sequence (seeing Table 2) that the HPV somatotype detects.

The HPV hypotype that table 1. detects and reaction reference

Table 2. primer sequence table

HPV hypotype/confidential reference items	Forward primer	Reverse primer
			6	GAGAAACATGGCGTACCGGAA	CTACCACCCAATCTGCACAT
11	CAGATCATCTGTAGCTAGTAGT	AAACTCCTCCACATGGCGCA
			16	GGAGGAGGATGAAATAGATG	GCTTTGTACGCACAACCGAA
18	GACGCAGAGAAACACAAGTA	CGGGCTGGTAAATGTTGAT
			26	CGGTAACAGTGGTATTTGATTG	CATTGCACACCTGTCCCATA

31	TCAAAGACCGTTGTGTCCAGA	GGGTTTCAGTACGAGGTCTT
			33	CTATGAGCAATTAAGTGACAGCT	TGTACTGTTGACACATAAACGA
35	AATTACAGCGGAGTGAGG	TGTCCACCGTCCACCGATGTTA
			39	TGGCCAATCGTGAAGGTACA	TGTCGCCACTGCTGTCT
42	ACGAACTAAGTCCTAGGCTT	CTACCACCCTTGTTGTAGGCGTA
			43	TAAGTGCCACAAGCCATTATCA	TTTCCAGCAATGTAAGCAG
44	TGCTATTTGTGCCACAAACCAT	GACCCTTCCAGGTATCTTGT
			45	GTATATGGAGAGACACTGGA	GCTATGCTGTGGAATCTTCGT
51	GGCTCCACCGTGCGCAGGGTC	AAACCGCAGCAGTGGC
			52	GAGCAATTAGGTGACAGCTC	AATGTGCCCAACAGCATCTGCT
53	ATGGATCGCCAGTTATTT	ACACAGCCAAGTTGCAGCTCCA
			56	GGTGCTACAGATGTCAAAGTC	GACCCGGTCCAACCATGTGCTAT
58	CCTGAACCAATTGACCTATTCT	GGTTGTTGTACTGTTGATACA
			59	GGACCCGAGCAAGACACCTAA	CTCGTAGCACACAAGGTCAAC
66	CCATCTGAGCGAGGTAT	TACCCTACATACTGCATATGGC
			68	CAGTACGTTTTAGCAGAGTAG	CCATAGGGTCAGACTGCT
73	CTGCAACTGTGGTGTAAGAT	AATGGCAAGGCATACTGT
			81	GGGCCAGCAAACCCTAC	GTCCGAAGCACGAATATTGC
82	GGCACCGGGTCTGGCACTG	CAAAGCCATCAGTACCAGTA
			83	GCTACGGCACTGGAGCCACTCA	TACCCTACACTGGCAGCA
beta-globin	CTCTTATCTTCCTCCCACAGCT	GACTTAGGGAACAAAGGAACCT
			pcDNA3.1(+)	CAGACAATCGGCTGCTCTGA	GCTTCAGTGACAACGTCGA

The implementation step:

1. uterine neck sample collection

Uterine neck bush, cell-preservation liquid

2. sample DNA extracts

Utilize paramagnetic particle method can extract simultaneously HPV and people's DNA.Ultimate principle is as follows:

6) get 500 μ L cell-preservation liquids in 1.5mL EP pipe, centrifugal 5 minutes of 13000g removes supernatant liquor, and the bottom is cervical exfoliated cell;

7) in the EP pipe that contains cervical exfoliated cell, add 500 μ L lysates, cracking 10 minutes;

8) in lysate, add the free DNA of 100 μ L magnetic beads absorption;

9) with scavenging solution magnetic bead is cleaned twice;

10) 100 μ L elutriant eluted dnas.

3. the DNA sample that extracts take step 2. carries out PCR as template and reacts

3.1 in 96 hole sample panel or eight connecting legs, add reagent and sample in following ratio:

3.2 carry out thermal cycle reaction by following temperature behind the mixing:

3.2GenomeLab GeXP genetic analyzer electrocapillary phoresis sample separation

3.3.1 preparation GeXP sample:

Annotate: PCR product amount can be 0.1-1 μ L, or water dilutes rear loading on demand in advance

3.3.2 preparation parting liquid: about 220 μ L parting liquids are added in the hole of proper number on the 96 hole parting liquid plates

3.3.3 electrocapillary phoresis sample separation.

3.3.4 interpretation of result.

4.HPV the somatotype immue quantitative detection reagent box comprises following reagent:

Primer pipe (Primer mix)

Solution X (Solution X)

PCR damping fluid (PCR Buffer)

25mM magnesium chloride (25mM MgCl ₂)

Polysaccharase (Taq polymerase)

Positive control (Positive control)

Obviously, the above embodiment of the present invention only is for example of the present invention clearly is described, and is not to be restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make on the basis of the above description other multi-form variation and changes.Here can't give all embodiments exhaustive.Everyly belong to the row that apparent variation that technical scheme of the present invention amplifies out or change still are in protection scope of the present invention.

Claims

1. the somatotype quantitative detecting method of a human papillomavirus (HPV) comprises the steps:

(1) collects the uterine neck sample;

(2) sample DNA extracts;

(3) carrying out PCR take the DNA sample that extracts as template reacts;

(4) with the method sample separation of electrocapillary phoresis.

2. detection method according to claim 1, described step (2) comprises following substep:

3) in lysate, add the free DNA of 100 μ L magnetic beads absorption;

4) with scavenging solution magnetic bead is cleaned twice;

5) 100 μ L elutriant eluted dnas.

3. detection method according to claim 1, described step (3) comprise the substep that carries out in proportion thermal cycle reaction after sample panel adds reagent and sample and mixing by certain temperature.

4. detection method according to claim 3, the thermal cycling temperature in the substep of described step (3) is respectively 94 ℃, 60 ℃, 70 ℃, 4 ℃.

5. detection method according to claim 1, described step (4) comprise preparation GeXP genetic analyzer sample, prepare the step of parting liquid, electrocapillary phoresis sample separation, interpretation of result.

6. a test kit that is used for each described method of aforementioned claim comprises following reagent: primer pipe, solution X, PCR buffer reagent, 25mM magnesium chloride, polysaccharase and positive control.